Hip fracture is a catalyst for profound morbidity and mortality in the aging population. Paradoxically, postmenopausal women with type 2 diabetes mellitus (T2DM) are at higher risk for hip fractures despite having higher bone mineral density than women without diabetes. The pathophysiology underlying this incongruity between bone quantity and quality with progressive glycemic derangement is unknown. In the aging skeleton, alterations in circulating factors have adverse effects on the bone microenvironment. Resident mesenchymal stem cells (MSCs) are the precursors to bone-building osteoblasts. In aging, they demonstrate decreased proliferation and preferential differentiation into adipocyte rather than osteoblast lineages. Despite the significant physiologic overlap in T2DM and aging, there has been little investigation into how common circulating factors have detrimental, local effects on diabetic bone and MSCs. The central hypothesis is that with progressive glycemic derangement, alterations in factors present in the circulation disrupt the bone microenvironment resulting in reduced MSC proliferation and differentiation, diminished matrix mineralization and impaired defense against oxidative stress consistent with premature skeletal aging. This hypothesis will be tested by pursuing three specific aims: 1) To determine the effect of serum from elderly subjects with either normal glucose tolerance (NGT), impaired glucose tolerance (IGT) or T2DM on human MSC (hMSC) proliferation, 2) To quantify the capacity of sera from elderly subjects with either NGT, IGT or T2DM to alter osteoblastic differentiation and matrix mineralization in hMSCs, and 3) To examine the induction of oxidative stress indices on undifferentiated hMSCs incubated with sera from these same subjects. For all experiments, human sera (HuS) from either young healthy female donors (30-45 years) or elderly donors (60 - 75 years) with NGT, impaired glucose tolerance (IGT) or T2DM (n=10 per group) will be incubated with an established line of hMSCs. To accomplish the first two aims, BrdU incorporation, TUNEL assay, osteoblast and adipocyte lineage allocation markers in quantitative real-time PCR (qRT-PCR) and cytochemical staining will be used to determine hMSC proliferation, differentiation and mineralization. To accomplish the third aim, reactive oxygen species and antioxidant enzyme expression and activity will be quantified using Fluorescence Activated Cell Sorting (FACS), qRT-PCR and biochemical assays. This approach is innovative because it 1) is founded on a novel model of T2DM as a precipitant of premature skeletal aging, and 2) tests this model by determining the effects of systemic factors in NGT, IGT and T2DM HuS on the MSC, an essential constituent of the marrow microenvironment. This research is significant because it will enhance understanding of the systemic pathways which prematurely age the diabetic skeleton. Greater knowledge of the mechanisms contributing to skeletal fragility in T2DM will translate into early interventions and targeted therapeutics to prevent future disability, morbidity and mortality following fracture. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because identifying the pathogenesis of skeletal failure in T2DM will speed the discovery of effective strategies for hip fracture prevention in the aging, diabetic population. This research is also expected to stimulate future exploration into the role of hormonal, metabolic and musculoskeletal dysregulation in the development of disability and frailty with age.